Abstract
Platelet adhesion to the sub-endothelial matrix and damaged endothelium occurs through a multi-step process mediated in the initial phase by glycoprotein Ib binding to von Willebrand factor, which leads to the subsequent formation of a platelet plug. The plant-derived omega-3 fatty acid alpha-linolenic acid is an abundant alternative to fish-derived n-3 FA and has anti-inflammatory and anti-thrombotic properties. In this study, we investigated the impact of alpha-linolenic acid on human platelet binding to vWF under high-shear flow conditions (mimicking blood flow in stenosed arteries). Pre-incubation of fresh human blood from healthy donors with alpha-linolenic acid at dietary relevant concentrations reduced platelet binding and rolling on vWF-coated microchannels at a shear rate of 100 dyn/cm2. Depletion of membrane cholesterol by incubation of platelet-rich-plasma with methyl-beta cyclodextrin abrogated platelet rolling on vWF. Analysis of glycoprotein Ib by applying cryo-electron tomography to intact platelets revealed local clusters of glycoprotein Ib complexes, upon shear-force exposure, whose formation could be prevented by alpha-linolenic acid treatment. This study provides novel findings on the rapid local rearrangement of the glycoprotein Ib complexes in response to high-shear flow and highlights the mechanism of in vitro inhibition of platelet binding to and rolling on vWF by alpha-linolenic acid.
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Key Points
The first event leading to the formation of a platelet plug is mediated by the glycoprotein Ib-IX complex (GpIb-IX), the second most-abundant platelet receptor after the integrin áIIbâ3). Platelet binding to von Willebrand factor (vWF) is tightly controlled in order to occur only at sites of bleeding but not in the normal circulation, where it would cause thrombosis. This regulation involves activation of vWF only at high flow rates and binding of GpIb to the A1 domain of vWF through a 2-step mechanism, where vWF multimer first elongates, then the A1 domain transitions to a high-affinity state. The role of high-shear flow in the pathogenesis of thrombosis is particularly relevant under pathological conditions such as in stenosed, atherosclerotic arteries, where shear stress can increase above 100 dyn/cm2 (shear rate > 4000/sec). Because of its pivotal role in initiating platelet adhesion, GpIb represents a promising anti-thrombotic target.
N-3 FA modulate cellular responses through incorporation into plasma membranes and reduction in the formation of typical protein complexes/lipid rafts, among other effects. ALA reduces platelet reactivity, and on studies showing the presence of GpIb in lipid rafts, it was hypothesized that ALA might interfere with GpIb distribution on the plasma membrane in high-shear flow and, therefore, binding to vWF.
The studies show ALA is able to partially inhibit platelet adhesion to vWF under a shear flow of 10’000, when whole blood is pre-incubated for 1 hour with ALA at dietary relevant concentrations.
The effect observed is specific to anchored vWF, since vWF binding upon exposure of platelet-rich plasm to high-shear flow could not be altered by ALA pretreatment.
The data provide insight into the possible mechanism of the anti-thrombotic properties of n-3 FA in the early phase of thrombosis at sites of arterial stenosis or plaque. It may therefore represent the basis for a therapeutic approach which interferes with this process.
In conclusion, the structural data from intact platelets, including cryo-ET and superresolution microscopy, show that upon high-shear conditions platelet GpIb receptors reorganize into “clusters” of 10 to 20 complexes closer to each other than on resting platelets; functionally, it was demonstrates that – by the intervention with the plant-derived n-3 FA ALA – the size of these clusters is reduced, thereby reducing platelet adhesion to vWF under high shear flow. This highlights ALA potential as an anti-thrombotic agent.